The present invention relates to commercial processes for the production of antiviral cis-1,3-oxathiolane and 1,3-dioxolane nucleoside analogues, some of which include, but are not limited to cis-(xe2x88x92)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (1, Lamivudine, 3TC, BCH-189) and its 5-fluoro analogue, cis-(xe2x88x92)-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (2, Emtricitabine, (xe2x88x92)-FTC)). In particular, the present invention relates to a new and efficient process for converting the undesired trans-1,3-oxathiolane and 1,3-dioxolane nucleoside isomers to the desired cis-1,3-oxathiolane and 1,3-dioxolane nucleoside isomers by a method of anomerization/transglycosylation, and a highly efficient method of separating the anomeric mixture of 1,3-oxathiolane nucleoside analogues to their single anomers. 
2,5-Disubstituted 1,3-oxathiolanes with pyrimidines or purine bases possess potent activities against the replication of the human immunodeficiency viruses (HIV) and hepatitis B virus (HBV). 3TC (1) has been approved as an antiviral for the treatment of HIV and HBV infection and its 5-fluoro analogue, (xe2x88x92)-FTC (2) is currently in advanced clinical trials for the same infections and shown promise as selective agent.
Generally speaking, there are two chiral centers in most of 1,3-oxathiolane and 1,3-dioxolane nucleosides and these nucleosides can exist in two distinct stereoisomeric forms, known as cis (xcex2) and trans (xcex1) diastereomers. Each cis- or trans-diastereomer is further composed of a pair of stereoisomers, known as enantiomers, and one of which displays high activity against viral infection. For many nucleoside analogues, including 3TC and FTC, the antiviral activity is significantly more pronounced in one of two possible enantiomeric forms of cis-diastereomer. In the case of 3TC and FTC, the levorotatory, or (xe2x88x92), enantiomer is the major contributor to the desired antiviral activity, as is disclosed in the following: U.S. Pat. No. 5,047,407; PCT WO 91/17159; U.S. Pat. No. 5,486,520; U.S. Pat. No. 5,539,116; J. Org. Chem. 1992, 57, 2217-2219; J. Med. Chem. 1993, 36, 181-195. Therefore, it would be very advantageous if these antiviral nucleoside analogues, especially 3TC and (xe2x88x92)-FTC, could be produced industrially in a straight forward and in a highly diastereoselective and enatioselective manner. Processes which would allow efficient preparation of pharmacologically desirable diastereomerically enriched cis-isomer, or, separation of cis/trans mixtures to provide diastereomerically enriched cis-isomer, or, efficient conversion into diastereomerically enriched cis-isomer from a mixture of trans-diastereomer and cis-diastereomer, are highly desirable.
Generally speaking there are two main strategies that have been used in the prior art for obtaining the desired enantiomer from the coupling of an oxathiolane derivative and a base. In the first strategy, the oxathiolane derivative is racemic and it is coupled to a base using various known coupling procedures giving only the racemic cis-isomer or a mixture of isomers (cis/trans) which is separated using known diastereomeric techniques to give the desired racemic cis-isomer. The racemic cis-isomer is resolved using enzymatic or chiral chromatography techniques (see for example EP 517145, PCT WO 00/09494). In the second strategy, single enantiomer or enantiomerically enriched 1,3-oxathiolane derivatives are coupled to a base to give a mixture of cis/trans-diastereomers, which can be separated using known diastereomeric techniques (see for example PCT W095/29174, PCT WO 00/09494).
The first strategy is not attractive because the resolution is performed at the end of the process and guarantees that 50% of the product would be the unwanted enantiomer which could not be easily converted to the desired enantiomer since two chiral centers would have to be inverted.
An advantage of the second strategy is that the resolution at the C-2 position of 1,3-oxathiolane derivatives occurs early in the process. Although an anomeric mixture may result where up to 50% of the product could be the unwanted trans-isomer, the possibility of converting the trans-isomer to the cis-isomer via anomerization/trans-glycosilation stands a much better chance of success than what would be needed in the first strategy since only one chiral center needs to be inverted. The second strategy could give a theoretical yield of 100% after the coupling step.
Nucleoside anomerization employing protic acids or Lewis acids has been applied to a wide variety of nucleosides and includes for example: 2M HCl, see F. Seela and H. D. Winkler, in xe2x80x9c2-Amino-7-xcex2-D-arabinofuranosyl-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine: a facile preparation and anomerization of a 7-deazapurine nucleosidexe2x80x9d (Carbohydrate Research, 1983, 118, 29-53); 1M HBr, see J. Cadet, Tetrahedron Lett., 1974, 867-870; NaI/HOAc, see J. Matulic-Adamic, et. al., in xe2x80x9cStereochemical features of the anomerizations in the 5,6-dihydrothymine nucleoside seriesxe2x80x9d (J. Chem. Soc. Perkin Trans. 1, 1988, 2681-2686); 2N HClO4, see J. Cadet and R. Teoule, in xe2x80x9cNucleic acid hydrolysis. I. Isomerization and anomerization of pyrimidic deoxyribonucleosides in an acidic mediumxe2x80x9d (J. Am. Chem. Soc., 1974, 96, 6517-6519); Ac2O/H2SO4, see R. T. Walker et. al., in xe2x80x9cA mild procedure for the anomerization of 2xe2x80x2-deoxynucleosidesxe2x80x9d (Tetrahedron Lett., 1993, 34, 6779-6782) and R. T. Walker et. al., in xe2x80x9cAnomerisation processxe2x80x9d (PCT WO 94/05686); Lewis acids, see D. Thacker and T. L. V. Ulbricht, in xe2x80x9cGeneral Lewis acid catalysis of glycoside anomerization and Oxe2x86x92N-glycosyl rearrangementxe2x80x9d (Chem. Commun. 1967, 122-123); AcOH, see L. N. Beigelman et. al., in xe2x80x9cEpimerization during the acetolysis of 3-O-acetyl-5-O-benzoyl-1,2-O-isopropylidene-3-C-methyl-xcex1-D-ribofuranose. Synthesis of 3xe2x80x2-C-methylnucleosides with the xcex2-D-ribo-and xcex1-D-arabino configurationsxe2x80x9d (Carbohydr. Res. 1988, 181, 77-88). This type of anomerization involves acid catalyzed sugar ring opening, forming a carbon cation at the anomeric carbon and cyclization to form the anomeric mixture of the nucleosides. This method is not suitable for 1,3-oxathiolane nucleoside analogues since the C-2 position of 1,3-oxathiolane ring could be epimerized under the reaction condition, which has been confirmed by Charron, et al. in xe2x80x9cRecycling of an undesired trans-[1,3]-oxathiolane nucleoside analogue by epimerizationxe2x80x9d (82nd Canadian Institute for Chemistry Conference, Organic Chemistry Abstract #530, May 30-Jun. 2, 1999, Toronto, Canada).
Base mediated anomerization has also been reported such as in, for example, 1:1 4N aqueous NaOH/Methanol; see V. W. Armstrong, et. al., in xe2x80x9cThe base catalysed anomerisation of xcex2-5-formyluridine; crystal and molecular structure of xcex1-formyluridinexe2x80x9d (Nucleic Acid Res., 1976, 3, 1791-1810); 2N aqueous NaOH, T. Ueda, et. al., in xe2x80x9cSynthesis of 5-alkyl-and 5-acyl-uridines via 6-mercaptouridine (nucleosides and nucleotides. XVII)xe2x80x9d (Heterocycles, 1977, 8, 427-432); anhydrous LiOH or KOH/MeOH, T. C. Britton et al., in xe2x80x9cProcess for anomerizing nucleosidesxe2x80x9d (EP 587364). This method may not be suitable for 1,3-oxathiolane systems since the 2-hydroxymethyl branch could also be epimerized. This comes about because the methine (C-2 position of 1,3-oxathiolane ring) proton on the oxo,thio acetal system is acidic, can be abstracted and thereby can lead to epimerization.
Nucleoside trans-glycosylation has been accomplished by the treatment of protected nucleoside with a base in the presence of a Lewis acid. The method has been applied for preparing anomeric mixture of the nucleosides, for example, H. Vorbruggen et. al. in xe2x80x9cNucleoside Synthesis, XXII. Nucleoside Synthesis with Trimethylsilyl Triflate and Perchlorate as Catalystsxe2x80x9d (Chem. Ber. 1981, 114 , 1234-1255) disclosed that a silylated xcex1-pyrimidinedione ribofuranosyl nucleoside was treated with TMS-triflate in acetonitrile to afford an xcex1-/xcex2-mixture (67/27) of that nucleoside. T. Yamaguchi and M. Saneyoshi in xe2x80x9cSynthetic Nucleosides and Nucleotides. XXI. On the synthesis and biological evaluations of 2xe2x80x2-deoxy-xcex1-D-ribofuranosy nucleosides and nucleotidesxe2x80x9d (Chem. Pharm. Bull., 1984, 32, 1441-1450) described that the fully acylated 2xe2x80x2-deoxycytidine was reacted with TMS-triflate and bis(trimethylsilyl)-acetamide (BSA) in dry acetonitrile at 70xc2x0 C. to give a mixture of cis-/trans-anomers. The reaction was believed to occur in two steps as an inter-molecular reaction. First, the trimethylsilylated base is released from the nucleoside and then the liberated active sugar carbonium cation at anomeric carbon position is attacked again by the nucleophilic center (for instance, N1-position of the pyrimidine base). This reaction can also be used for preparing a new nucleoside by employing a new base. M. Miyaki et. al. in xe2x80x9cNxe2x86x92N Alkyl and glycosyl migrations of purines and pyrimidines. IV. Trans-Glycosylation from pyrimidines to purines. (A novel synthetic method of purine nucleosides and nucleotides)xe2x80x9d (Chem. Pharm. Bull., 1970, 2459-2468) described that trans-glycosylation from pyrimidines to purines catalyzed by Lewis acid such as HgBr2 and SnCl4. M. Imazawa and F. Eckstein in xe2x80x9cSynthesis of 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxyribofuranosylpurinesxe2x80x9d (J. Org. Chem. 1978, 43, 3044-3048) described that the trans-glycosylation reaction of 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-deoxy-5xe2x80x2-O-acetylthymidine with silylated N6-octanoyladenine using trimethylsilyl trifluoromethanesulfonate as a catalyst afforded a mixture of xcex1 and xcex2 anomers of 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxyadenosine. Such examples also can be found in the following articles: B. Shimizu and M. Miyaki in xe2x80x9cTransglycosylation from pyrimidines to purinesxe2x80x9d (Tetrahedron Lett., 1968, 855-859); T. Azuma, et. al. in xe2x80x9cChemical transglycosylation of octosyl acidxe2x80x9d (Tetrahedron Lett., 1976, 1687-1690); T. Azuma, et. al. in xe2x80x9cTransglycosylation: an improved method for tansglycosylation from pyrimidines to purinesxe2x80x9d (Chem. Pharm. Bull. 1977, 25, 3347-3353); M. Imazawa and F. Eckstein in xe2x80x9cFacile Synthesis of 2xe2x80x2-Amino-2xe2x80x2-deoxyribofuranosyl Purinesxe2x80x9d (J. Org. Chem. 1979, 44, 2039-2041); J. Kiss, et. al. in xe2x80x9cStereospecific synthesis of the anticancer agent 5xe2x80x2-deoxy-5-fluorouridine (5-DFUR) and its 5xe2x80x2-deuterated derivativesxe2x80x9d (Helv. Chim. Acta 1982, 65, 1522-37); A. V. Azhayev et. al. in xe2x80x9cAminonucleosides and their derivatives; XIII. Synthesis of Benzimidazole azidonucleosidesxe2x80x9d (Synthesis, 1985, 410-411); S. L. Beaucage, et. al., in xe2x80x9cSynthesis and physicochemical properties of alternating xcex1,xcex2-oligodeoxyribonucleotides with alternating (3xe2x80x2xe2x86x923xe2x80x2)- and (5xe2x80x2xe2x86x925xe2x80x2)-internucleotidic phosphodiester linkagesxe2x80x9d (J. Org. Chem. 1995, 60, 1520-1530); K. Pongracz and S. M. Gryaznov, in xe2x80x9cxcex1-Oligodexyribonucleotide N3xe2x80x2xe2x86x92N5xe2x80x2 phosphoramidates: synthesis and duplex formationxe2x80x9d (Nucleic Acids Research, 1998, 26, 1099-1106); M.-C. Liu, et. al. in xe2x80x9cSynthesis and Biological Evaluation of 1,3-Oxathiolane 5-Azapyrimidine, 6-Azapyrimidine, and Fluorosubstituted 3-Deazapyrimidine Nucleosidesxe2x80x9d (Nucleosides, Nucleotides and Nucleic Acids, 2000, 19, 603-618).
D. A. Carson and D. Bruce Wasson in xe2x80x9cSynthesis of 2xe2x80x2,3xe2x80x2-dideoxylnucleosides by enzymatic trans-glycosylationxe2x80x9d (Biochem. Biophys. Res. Commun. 1988, 155, 829-834) disclosed that 2xe2x80x2,3xe2x80x2-dideoxynucleosides could be prepared by enzymatic trans-glycosylation using the trans-N-deoxyribosylase from Lactobacillus helveticus. 
Although anomerization/epimerization and trans-glycosylation of general nucleosides have been well studied, efficient anomerization and trans-glycosylation reactions for converting pharmacologically undesired trans-1,3-oxathiolane and trans-1,3-dioxolane nucleosides into their pharmacologically desired cis-isomers or cis-/trans-isomers mixture have not been explored.
The current processes for the separation of cis-/trans-isomers of 1,3-oxathiolane and 1,3-dioxolane nucleosides are also limited. They are often separated by physical means e.g. chromatographic method (L. S. Jeong, et. al. in xe2x80x9cAsymmetric Synthesis and Biological Evaluation of xcex2-L-(2R,5S)- and xcex1-L-(2R, 5R)-1,3-Oxathiolane-Pyrimidine and purine Nucleosides as potential anti-HIV agentsxe2x80x9d (J. Med. Chem. 1993, 36, 181-195); D. C. Humber, et. al. in xe2x80x9cExpeditious preparation of (xe2x88x92)-2xe2x80x2-deoxy-3xe2x80x2-thiacytidine (3TC)xe2x80x9d (Tetrahedron Lett. 1992, 33, 4625-4628)) or by crystallization method (in xe2x80x9cProcess for the diastereoselective synthesis of nucleoside analoguesxe2x80x9d (WO 95/29174), in xe2x80x9cProcesses for the diastereoselective synthesis of nucleosidesxe2x80x9d (EP 0 515 157 A1), and in xe2x80x9cMethod of manufacture of 1,3-oxathiolane nucleosidesxe2x80x9d (WO 00/09494). These processes started from the nucleosides with chiral auxiliary or the diastereomeric enriched nucleosides mixture). As can be seen, an efficient and straightforward method for separating cis-/trans-isomers mixture of 1,3-oxathiolane and 1,3-dioxolane nucleosides, especially 1:1 cis-/trans-isomers mixture, still need to be developed.
It is therefore an object of the present invention to provide a commercial process for the conversion of pharmacologically undesired trans-1,3-oxathiolane and 1,3-dioxolane nucleoside analogues into the desired cis-form. In particular, according to one aspect of the invention, a process is provided for increasing the amount of cis-nucleoside isomer of the formula 
in a mixture of cis-nucleoside and trans-nucleoside isomers, wherein R1 is selected from the group consisting of hydrogen, lower alkyl, fluoro, azide, hydroxy, and OA where A is a lower alkyl, aryl, alkylsilyl, and acyl groups; R2 is selected from the group consisting of hydrogen, azide, lower alkyl, fluoro, hydroxy (provided R3 cannot be fluoro, azide, or hydroxy), and OA where A is as defined above; R3 is selected from the group consisting of hydrogen, azide, lower alkyl, fluoro, hydroxy (provided R2 cannot be fluoro, azide, or hydroxy), and OA where A is as defined above; X and Y are independently selected from the group consisting of oxygen and sulfur; and B is a pyrimidine or purine base, which includes, but is not limited to, 6-alkylpurine and N6-alkylpurines, N6-acylpurines, N6-benzylpurine, 6-halopurine, N6-acetylenic purine, N6-acyl purine, N6-hydroxyalkyl purine, 6-thioalkyl purine, N2-alkylpurines, N4-alkylpyrimidines, N4-acylpyrimidines, 4-halopyrimidines, N4-acetylenic pyrimidines, 4-amino and N4-acyl pyrimidines, 4-hydroxyalkyl pyrimidines, 4-thioalkyl pyrimidines, thymine, cytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrimidine, uracil, C5-alkylpyrimidines, C5-benzylpyrimidines, C5-halopyrimidines, C5-vinylpyrimidine, C5-acetylenic pyrimidine, C5-acyl pyrimidine, C5-hydroxyalkyl purine, C5-amidopyrimidine, C5-cyanopyrimidine, C5-nitropyrimidine, C5-aminopyrimidine, N2-alkylpurines, N2-alkyl-6-thiopurines, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethyl-t-hexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl, propionyl and butyryl, methanesulfonyl, and p-toluenesulfonyl. Preferred bases include cytosine, 5-fluorocytosine, uracil, thymine, adenine, guanine, xanthine, 2,6-diaminopurine, 6-aminopurine, 6-chloropurine and 2,6-dichloropurine.
Surprisingly we have discovered a novel process for converting the undesired trans-1,3-oxathiolane and 1,3-dioxolane nucleoside analogues, in their protected or unprotected forms into a mixture of the desired cis- and trans- isomers by trans-glycosylation such as, for example,
(i) by treating the undesired trans nucleoside with a suitable acid and a purine or pyrimidine base, or a derivative thereof, or
(ii) by treating the undesired trans nucleoside with a suitable silylation reagent (such as BSA which is an anacronym for (bis-trimethyl-silyl) acetamide) and a suitable acid such as a Lewis acid in the presence or in the absence of a nucleoside base, to provide the mixture of cis-/trans-isomers. The mixture of cis-/trans-isomers, if necessary, can be separated such as via the crystallization of them or their organic or inorganic acid salts, for example, according to a procedure we disclose below.
It is also an object of this invention to provide a process for the efficient separation of desired cis-1,3-oxathiolane and 1,3-dioxolane nucleoside analogues of formula (3) from the mixtures of cis-/trans-isomers. In accordance with an aspect of the invention, a process is provided for the separation of the anomeric mixture of 1,3-oxathiolane and 1,3-dioxolane nucleosides, comprising the use of pyrimidine or purine base. Another aspect of the invention also provides a process for separating them in their optically active forms. The anomeric mixture may be, for example, treated with a suitable acid, and the corresponding salts of isomers are separated in their cis- or trans-form in high optical purity and high chemical yield by a method of fractional crystallization. The pharmacologically desired cis-isomer salt is subject to free basing to offer cis-1,3-oxathiolane or 1,3-dioxolane nucleoside. The undesired trans isomers can also be recycled and subjected to anomerization/trans-glycosylation to re-form desired cis-isomers in accordance with aspects of the invention.
In aspects of the present invention, the undesired 1,3-oxathiolane nucleosides trans-isomers (compound 4), in their protected or unprotected form, may be subjected to anomerization/trans-glycosylation in the presence of acids and a pyrimidine or purine base in a single solvent or solvent mixture, to give the desired cis-1,3-oxathiolane nucleoside analogues or the mixture of cis- and trans-isomers (5). 
Scheme 1 depicts such a process, wherein R1, R2, R5 are independently selected from the group consisting of hydrogen, alkyl, alkylsilyl, aryl and acyl groups and R3 and R4 are independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, low alkyl, aryl, amino and hydroxyl groups.
The nucleosides, which are used as the starting material, can be single trans-isomers or trans-/cis-isomer mixtures, for example, predominantly as the trans-isomer.
Examples of the preferable 1,3-oxathiolane nucleosides produced are, for instance, cis-(xe2x88x92)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (1, 3TC), cis-(xe2x88x92)-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (2, FTC), and the like.
The acids which can be used for this process, include, but are not limited to, chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane, trimethylsilyl trifluoromethanesulfonate, trimethylsilyl trifluoroacetate, triethylsilyl trifluoromethanesulfonate, t-butyldimethylsilyl chloride, t-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, boron trichloride, boron trifluoride, boron tribromide, boron triiodide, boron trifluoroacetate, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid. The preferable acids are, for instance, iodotrimethylsilane, trimethylsilyl trifluoromethanesulfonate, p-toluenesulfonic acid, and the like.
The amount of the acid can range from 0.05 to 5 moles per mole of the nucleoside. The preferable amount of the acid is from 1.0 to 2.0 moles per mole of the nucleoside.
The pyrimidine or purine bases can be used as is or in their protected forms. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethyl-t-hexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl, propionyl and butyryl, methanesulfonyl, and p-toluenesulfonyl. Preferred bases include cytosine, 5-fluorocytosine, and the like.
The amount of the pyrimidine bases can range from 0 to 5 moles per mole of the nucleoside. The preferable amount is from 0.5 to 1.5 moles per mole of the nucleoside.
Examples of the preferable solvents for trans-glycosylation/anomerization are, for instance, toluene, benzene, xylenes, dichloromethane, dichloroethanes, chloroform, acetonitrile, tetrahydrofuran, ethers, 1,4-dioxane, etc., and their mixtures.
The reaction temperature preferably ranges from xe2x88x9225xc2x0 C. to 150xc2x0 C. The preferable reaction temperature is from 0 to 50xc2x0 C.
The overall chemical yield of the trans-glycosylation/anomerization generally ranges from 30% to 99%.
The ratio of the product of cis-/trans-nucleosides mixture ranges from 1:5 (cis/trans) to 1:0(cis/trans) after anomerization. The most common ratio is from 1:2 (cis/trans) to 2:1(cis/trans).
After trans-glycosylation/anomerization, the protected nucleosides mixture is subjected to de-protection by using known procedures if necessary, for instance, base mediated deacylation, and the like. The cis-/trans-nucleosides mixture can be separated by the fractional crystallization of their salts of an inorganic or organic acid, by employing the following procedure.
As shown in scheme 2, the mixture of cis-/trans-isomers of 1,3-oxathiolane nucleoside analogues (6) is treated with inorganic or organic acids in a single solvent or solvent mixture; the corresponding salts of isomers are separated in high optical purity by fractional crystallization. The desired cis-nucleoside salts (7) are isolated in high optical purity and high chemical yields. These salts are converted into their corresponding free base form by treatment with organic or inorganic bases in a single solvent or a solvent mixture in high chemical yields. The undesired trans-nucleoside salts (8) are also isolated in high optical purity and chemical yield and they can be recycled by following the process we disclosed in the present invention. 
wherein R1 may be hydrogen, low alkyl, aryl, alkylsilyl, and acyl groups. R2 may be hydrogen, alkyl, aryl, and alkylsilyl acyl groups. R3 and R4 are independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, low alkyl, aryl, amino and hydroxyl groups.
Examples of the preferable 1,3-oxathiolane nucleosides produced are, for instance, cis-(xe2x88x92)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (1, 3TC), cis-(xe2x88x92)-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (2, FTC), and the like.
Examples of the inorganic and organic acids which can be used for the salt formation and separation, include, but are not limited to, methanesulfonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid. The preferable acids are methanesulfonic acid, hydrochloric acid, and the like. The process may use a single acid, or use a combination of several acids.
The amount of the acid can range from 0.5 to 5 moles per mole of the nucleoside. The preferable amount of the acid is from 1.0 mole to 1.5 moles per mole of the nucleoside.
Examples of the preferable solvents for fractional crystallization can be protic or aprotic such as methanol, ethanol, propanols, butanols, ethers, ethyl acetate, water, etc., and their mixtures.
The overall chemical yield for separating the two isomers ranges from 30 to 99%.
Examples of the preferable organic or inorganic bases for free basing step are, for instance, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, triethylamine, ammonium hydroxide, ammonia, basic resins, and the like. The process may use a single base, or use a combination of several bases.
Examples of the preferable solvents for the free basing step are, for instance, methanol, ethanol, propanols, butanols, ethers, ethyl acetate, water, etc., and their mixtures.
Thus, in accordance with an aspect of the invention, a process is provided for the preparation of 1,3-oxathiolane nucleoside analogues in predominantly the cis-form, from mixture of cis-/trans-1,3-oxathiolane nucleosides or their protected derivatives thereof, comprising:
(i) treatment of the cis-/trans-1,3-oxathiolane nucleosides (or protected derivatives thereof) with a pyrimidine base (or it derivatives thereof) and an acid,
(ii) adding a suitable acid to the obtained cis-/trans-mixture of isomers,
(iii) selective crystallization of the desired cis-isomer salt from a solvent or combination of solvents, and
(iv) treatment of the predominantly cis-isomer salt with a suitable base to offer the free 1,3-oxathiolane nucleosides, and thereafter optionally repeating steps (i) to (iv) inclusive.
According to an aspect of the invention, the 1,3-oxathiolane nucleoside provided may be cis-(xe2x88x92)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (Lamivudine, 3TC).
Further, according to another aspect of the invention, the 1,3-oxathiolane nucleoside produced is cis-(xe2x88x92)-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (Emtricitabine, (xe2x88x92)-FTC).
Further, according to another aspect of the invention, the pyrimidine base is N,O-disilyled cytosine.
Further, according to another aspect of the invention, the pyrimidine base is N,O-disilyled 5-fluoro-cytosine.
Further, according to another aspect of the invention, the acid for anomerization/trans-glycosylation is iodotrimethylsilane or trimethylsilyl trifluoromethanesulfonate.
Further, according to another aspect of the invention, the acid used for salt formation is methanesulfonic acid or hydrochloric acid.
Further, according to another aspect of the invention, the solvent for crystallization is ethanol, methanol, and ethyl acetate.
Further, according to another aspect of the invention, cis-(xe2x88x92)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone, mesylate is provided.
Further, according to another aspect of the invention, cis-(xe2x88x92)-Amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone, mesylate is provided.
Further, according to another aspect of the invention, a process is provided for the conversion of pharmacologically undesired trans-1,3-oxathiolane and 1,3-dioxolane nucleoside analogues into the desired cis-form thereby increasing the amount of cis-nucleoside isomer of the formula 
in a mixture of cis-nucleoside and trans-nucleoside isomers is provided, wherein R1 is selected from the group consisting of hydrogen, lower alkyl, fluoro, azide, hydroxy, and OA where A is a lower alkyl, aryl, alkylsilyl, and acyl groups; R2 is selected from the group consisting of hydrogen, azide, lower alkyl, fluoro, hydroxy (provided R3 cannot be fluoro, azide, or hydroxy), and OA where A is as defined above; R3 is selected from the group consisting of hydrogen, azide, lower alkyl, fluoro, hydroxy (provided R2 cannot be fluoro, azide, or hydroxy), and OA where A is as defined above; X and Y are independently selected from the group consisting of oxygen and sulfur; and B is a pyrimidine or purine base, which includes, but is not limited to, 6-alkylpurine and N6-alkylpurines, N6-acylpurines, N6-benzylpurine, 6-halopurine, N6-acetylenic purine, N6-acyl purine, N6-hydroxyalkyl purine, 6-thioalkyl purine, N2-alkylpurines, N4-alkylpyrimidines, N4-acylpyrimidines, 4-halopyrimidines, N4-acetylenic pyrimidines, 4-amino and N4-acyl pyrimidines, 4-hydroxyalkyl pyrimidines, 4-thioalkyl pyrimidines, thymine, cytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrimidine, uracil, C5-alkylpyrimidines, C5-benzylpyrimidines, C5-halopyrimidines, C5-vinylpyrimidine, C5-acetylenic pyrimidine, C5-acyl pyrimidine, C5-hydroxyalkyl purine, C5-amidopyrimidine, C5-cyanopyrimidine, C5-nitropyrimidine, C5-aminopyrimidine, N2-alkylpurines, N2-alkyl-6-thiopurines, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl and wherein functional oxygen and nitrogen groups on the base can be protected as necessary or desired by suitable protecting groups which may include trimethylsily, dimethyl-t-hexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl, propionyl and butyryl, methanesulfonyl, and p-toluenesulfonyl by trans-glycosilation,
(i) by treating the undesired trans nucleoside with a suitable acid and a suitable purine or pyrimidine base, or a derivative thereof, or
(ii) by treating the undesired trans nucleoside with a suitable silylation reagent (such as BSA (bis-trimethyl silyl) acetamide) and a suitable acid such as a Lewis acid in the presence or in the absence of a nucleoside base, to provide the mixture of cis-/trans-isomers.
Further, according to another aspect of the invention, the process is carried out by carrying out the process of subparagraph (i) in the immediately preceding paragraph.
Further, according to another aspect of the invention, the process is carried out by carrying out the process of subparagraph (ii) above in the next to the immediately preceding paragraph.
Further, according to another aspect of the invention, the mixture of cis-/trans-isomers can be separated via the crystallization of them or their organic or inorganic acid salts.
Further, according to another aspect of the invention, the pharmacologically desired cis-isomer salt is subject to free basing to offer cis-1,3-oxathiolane or 1,3-dioxolane nucleoside and wherein the undesired trans compounds are optionally recycled and subjected to anomerization/trans-glycosylation to re-form desired cis-isomers.
Further, according to another aspect of the invention, a process is provided for converting 1,3-oxathiolane nucleosides trans-isomers (compound 4), 
in their protected or unprotected form, the process comprising subjecting the trans-isomers to anomerization/trans-glycosylation in the presence of acids and a suitable pyrimidine or purine base in a single solvent or solvent mixture, to give the desired cis-1,3-oxathiolane nucleoside analogues or the mixture of cis- and trans-isomers (5) 
Further, according to another aspect of the invention, R1, R2, R5 are independently selected from the group consisting of hydrogen, alkyl, alkylsilyl, aryl and acyl groups. R3 and R4 are independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, low alkyl, aryl, amino and hydroxyl groups.
Further, according to another aspect of the invention, the nucleosides, which are used as the starting material, are selected from single trans-isomers or trans-/cis-isomer mixtures predominantly containing the trans-isomer.
Further, according to another aspect of the invention, the 1,3-oxathiolane nucleosides produced are selected from cis-(xe2x88x92)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (1, 3TC), cis-(xe2x88x92)-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone (2, FTC), and the like.
Further, according to another aspect of the invention, the acids are selected from chlorotrimethylsilane, bromotrimethylsilane, iodotrimethylsilane, trimethylsilyl trifluoromethanesulfonate, trimethylsilyl trifluoroacetate, triethylsilyl trifluoromethanesulfonate, t-butyldimethylsilyl chloride, t-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, boron trichloride, boron trifluoride, boron tribromide, boron triiodide, boron trifluoroacetate, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid.
Further, according to another aspect of the invention, the acids are selected from iodotrimethylsilane, trimethylsilyl trifluoromethanesulfonate, and the like.
Further, according to another aspect of the invention, the amount of the acid ranges from 0.05 to 5 moles per mole of the nucleoside.
Further, according to another aspect of the invention, the amount of the acid is from 1.0 to 2.0 moles per mole of the nucleoside.
Further, according to another aspect of the invention, the pyrimidine or purine bases are selected from pyrimidine and purine bases protected by trimethylsily, dimethyl-t-hexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl, propionyl and butyryl, methanesulfonyl, and p-toluenesulfonyl.
Further, according to another aspect of the invention, the bases are selected from cytosine, 5-fluorocytosine, and the like.
Further, according to another aspect of the invention, the amount of the pyrimidine bases can range from 0 to 5 moles per mole of the nucleoside.
Further, according to another aspect of the invention, the amount of base is from 0.5 to 1.5 moles per mole of the nucleoside.
Further, according to another aspect of the invention, a process is provided comprising treating a mixture of cis-/trans-isomers of 1,3-oxathiolane nucleoside analogues (6) 
with inorganic or organic acids in a single solvent or solvent mixture; the corresponding salts of the isomers being thereafter separated in high optical purity by fractional crystallization, the desired cis-nucleoside salts (7) 
being isolated and converted into their corresponding free base form (9) by treatment with organic or inorganic bases in a single solvent or a solvent mixture, the undesired trans-nucleoside salts (8) 
also being isolated and recycled by repeating the process thereby to produce 
wherein R1 is selected from hydrogen, low alkyl, aryl, alkylsilyl, and acyl groups, R2 selected from hydrogen, alkyl, aryl, and alkylsilyl groups and R3 and R4 are independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, low alkyl, aryl, amino and hydroxyl groups.
Further, according to another aspect of the invention, R3xe2x95x90R4=H in compound 9 and compound 9 is 3TC.
Further, according to another aspect of the invention, R3 is F and R4 is H in compound 9 and compound 9 is FTC.
Further, according to another aspect of the invention, the inorganic and organic acids are selected from methanesulfonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid.
Further, according to another aspect of the invention, the acid is selected from methanesulfonic acid, hydrochloric acid, and the like.
Further, according to another aspect of the invention, the amount of the acid can range from 0.5 to 5 moles per mole of the nucleoside.
Further, according to another aspect of the invention, the amount of the acid is from 1.0 mole to 1.5 moles per mole of the nucleoside.